A common hallmark of metastasis is a loss of expression of the E-cadherin cell adhesion protein, which is associated with the epithelial mesenchymal transition in the tumor microenvironment. However, many metastases still contain high levels of E-cadherin expression and epithelial cells expressing E-cadherin can become invasive and metastasize. We have discovered that E-cadherin adhesive activity is dynamically regulated at the cell surface in tumor cells and have developed ways to strongly activate its adhesive function using a unique monoclonal antibodies (mAbs) as well as specific mutations in the associated p120-catenin. Preliminary findings demonstrate that activating mAbs slow mammary tumor metastasis to lungs in a mouse model. Additional findings show that human cancer promoting mutations in E-cadherin affect this activation process specifically but not its basic adhesive function. In another line of research, we have discovered that E-cadherin stimulates the Hippo signaling pathway, which mediates contact inhibition of growth and may also confer chemoresistance in late stage tumors. Preliminary experiments indicate that activating mAbs enhance Hippo signaling in cell cultures at contact free edges of the colony. I hypothesize that progression and metastasis of E-cadherin positive tumors is controlled by the activity state of E-cadherin at the cell surface through its adhesion and/or signaling functions; and that reduced functional activity of E-cadherin at the cell surface results from either E-cadherin mutations or altered responses to micro-environmental factors. The overall objective is to test this hypothesis by determining the effects of activating E-cadherin on the growth and metastasis of mammary tumor cells, and by determining whether cancer associated mutations in E-cadherin affect its activity state and/or ability to signal to the growth inhibitory Hippo signaling pathway. The specific aims are to: A. Investigate how E- cadherin activation inhibits the metastasis of mammary tumors, by exploring various ways of inducing E- cadherin activation in different tumor contexts, and by determining the stage at which the metastatic process in affected. B. Investigate whether E-cadherin missense mutations associated with human cancer interfere with normal mechanisms of activation and how they affect metastasis, using in vitro assays and in vivo models, respectively. C. Investigate how E-cadherin activation and cancer-associated E-cadherin mutations affect Hippo signaling in tumor cells, in vivo and in vitro, and whether regulation of the Hippo signaling pathway is involved in E-cadherin regulation of mammary tumor metastasis. The main purpose of these proposed studies is to understand principles of microenvironmental regulation of E-cadherin function in metastasis, but investigating the actions of the activating mAbs could lead to adjunct therapies for metastasis and associated chemoresistance.